Title: pGLO Transformation Lab Introduction: Genetic transformation is a change caused by genes, involving the insertion of a gene into

an organism to change the organisms trait. In this experiment, bacteria will be transformed with a gene that codes for Green Fluorescent Protein (GFP). The bacteria with this gene will cause them to glow a brilliant green color under ultraviolet light. Plasmid DNA contains genes for one or more traits that may be beneficial to survival. In this case, the pGLO is resistant to the antibiotic ampicillin. The gene for GFP can be switched on in transformed cells by adding the sugar arabinose to the cells nutrient medium. Transformed cells will grow on plates with LB/amp, and appear white on plates not containing arabinose. Hypothesis: If bacteria with +pGLO plasmids that are resistant to the antibiotic ampicillin and have the gene for GFP, colonies will survive and grow on the transformation plates that have LB/amp. In addition, +pGLO bacteria on a plate with LB/amp/ara will grow and glow green under UV light because of the inclusion of arabinose. In the control plates, -pGLO bacteria that are amp sensitive will not be able to grow on the LB/amp plates. The other control plate with pGLO bacteria and no ampicillin added will host a lawn of colonies. Materials: Starter plate with the starting bacteria, 4 micro test tubes, foam tube rack, 500 l CaCl2, sterile pipets, sterile loops, 500 l LB nutrient broth, rack on ice, 4 poured agar plates, and hot water bath at 42C are needed. Procedure: To do a transformation, one micro test tube was labeled +pGLO and another pGLO and placed in a foam tube rack. The tubes were opened and 250l of transformation solution (CaCl2) was transferred using a sterile transfer pipet. The tubes were placed on ice. Using a sterile loop, a single colony of bacteria was picked up from the starter plate. The loop was immersed into the +pGLO tube. The tube was placed back in the tube rack in the ice. This was repeated for the pGLO tube. Plasmid DNA was added into the +pGLO tube and returned back to the ice rack. Plasmid was not added to the pGLO tube. The tubes were incubated on ice for 10 minutes, with the tubes pushed all the way down in the rack so they made contact with the ice. When the tubes are sitting on ice, four agar plates were labeled as follows: LB/amp +pGLO, LB/amp/ara +pGLO, LB/amp pGLO, and LB pGLO. Then the foam rack with the tubes were heat shocked in a water bath set at 42C for exactly 50 seconds. When the 50 seconds were done, both tubes were placed back on ice with a rapid change. The tubes were incubated for 2 minutes on ice. The rack was removed from the ice. Using a new sterile pipet, 250 l of LB nutrient broth was added to each tube. The tubes were incubated at room temperature for 10 minutes. Then, using a sterile loop for each tube, 100 l were pipetted into the appropriate plates. Using a new sterile loop for each plate, the suspensions were spread evenly around the surface of the agar. Then the plates were stacked up, taped together, placed upside down, and left at room temperature for 3 days instead of being incubated at 37C.

Results: Class Transformation Efficiency (Transformants/g) Group 1 Efficiency 38.22 Group 3 Calculations: Total number of fluorescent cells: 633 Total amount of pGLO plasmid DNA used (DNA g) = (Concentration of DNA in g/l) x (volume of DNA in l) .157 g Transformation efficiency = (total # of cells growing on the agar plate)/(Amt of DNA spread on agar plate) 4,031.85 transformants/g 2 6.36 3 4 4,031.85 31.847 5 216.56 6 44.6 7 31.847 8 1,547.8

Transformation plates: LB/amp had 771 colonies and appeared white. LB/amp/ara had 633 colonies, appeared white in room light, and glowed green under UV light. Control plates: LB/amp had no growth. LB had a white lawn of bacteria. Lesson 1 Focus Questions 1. Which organism is better suited for total genetic transformation-one composed of many cells, or one composed of a single cell? An organism composed of a single cell is better for genetic transformation, because that one cell can take up the new gene. 2. Scientists often want to know if the genetically transformed organism can pass its new traits on to its offspring and future generations. To get this information, which would be a better candidate for your investigation, an organism in which each new generation develops and reproduces quickly, or one which does this more slowly? An organism which reproduces quickly, like bacteria.

3. What traits should the organism have (or not have) to be sure it will not harm you or the environment? The organism shouldnt be toxic towards humans, plants, or animals. It should grow well in a lab, but shouldnt grow outside the lab. 4. Which would be the best choice for a genetic transformation: a bacterium, earthworm, fish, or mouse? Bacterium, because they are single celled, small, and reproduces quickly. But the bacteria cant harm people or survive outside the lab. Lesson 2 Review Questions 1. On which of the plates would you expect to find bacteria most like the original untransformed E. coli colonies you initially observed? On the control plates (pGLO/LB) because these bacteria were removed from the starter plate and didnt have plasmids added. 2. If there are any genetically transformed bacterial cells, on which plate(s) would they most likely be located? On the LB/amp and LB/amp/ara plates. Genetically transformed cells can survive on the ampicillin plates. 3. Which plates should be compared to determine if any genetic transformation has occurred? The LB/amp pGLO and LB/amp +pGLO should be compared. Cells without pGLO wont grow on the amp plates, and cells with it will grow. 4. What is meant by a control plate? What purpose does a control serve? The control plates are the pGLO plates, so that the plates can be compared to see if the bacteria were really transformants. The pGLO LB/amp control shows the starter culture doesnt grow on LB/amp. Lesson 3 Review Questions Do you observe some E. Coli growing on the LB plates which do not contain ampicillin/arabinose? Yes, the bacteria without plasmids in the plain LB plate (lawn). 1. From your results, can you tell if these bacteria are ampicillin resistant by looking at them on the LB plate? No, because the amp resistant and amp sensitive look similar when cultured. 2. How would you change the bacterias environment to best tell if they are amp resistant? If you take the bacteria on the LB plate and put them on a LB/amp plate, they are resistant to amp if they survive. If they dont survive, then the bacteria wasnt amp resistant. 3. A) What two factors must be present in the bacterias environment for you to see the green color? The arabinose in the agar plate turns on the GFP gene. The UV light helps fluoresce the protein. B) What do you think each of the two environmental factors listed above are doing to cause the genetically transformed bacteria turn green? The arabinose turns on

the GFP gene by binding to a protein. Then the gene is transcribed. The UV light causes the GFP to look green. C) What advantage would there be for an organism to be able to turn on or off particular genes in response to certain conditions? The organism can adapt to conditions and wont have to waste and spend energy producing unnecessary proteins. Sources of Error: If correct lab technique wasnt used, unwanted microbes would be in the plates when they were open for two long. Then the data wont be reliable. Also, if the incubation time wasnt exact, thats also room for error. Discussion and Conclusion: This experiment was successful in determining the transformation efficiency and results of genetic transformation. The plasmid must have given resistance to the antibiotic on the plates that contained ampicillin. The pGLO bacteria that didnt have the plasmid couldnt survive on the ampicillin plates, which resulted in no growth of the control. Another plate of pGLO thrived in a plate that didnt contain the antibiotic. The two plates that contained the plasmids both had bacterial resistance to the antibiotic. In addition, the plate that had LB/ampicillin/arabinose gave the colonies a fluorescent green glow under ultraviolet light. Transformation efficiency is the quantitative value that describes how effective the transfer of plasmids into bacteria. The number represents the number of transformed colonies produced per microgram of DNA added. Most groups had ideal transformation efficiencies, but some groups did not reach the optimum number (i.e. group 2 with 6.36 transformants/g).